Project Summary Production of hyperconcentrated (dehydrated) mucus is a common feature of chronic obstructive pulmonary disease (COPD), including chronic bronchitis (CB). Mucus hydration is driven by electrolyte transport activities, which in turn are regulated by airway epithelial purinergic receptors. The activity of these receptors is controlled by the extracellular concentrations of ATP and its metabolite adenosine (Ado). The objective of this proposal is to elucidate the contribution of nucleotide release and metabolism to mucin hydration and clearance in health and CB-diseased airways. Our preliminary data leads us to hypothesize that deficient ATP/ Ado levels in airway surface liquid (ASL) lead to reduced mucin hydration and increased plug formation, and contribute to CB disease pathogeneses. To test this hypothesis, we will first (Aim 1) measure the rates of ATP release and metabolism in primary cultures of human bronchial epithelial (HBE) cells under normal conditions and in cultures exposed to conditions relevant to CB: a) Th1 cytokine-induced goblet cell hyperplasia; and b) cigarette smoke (CS). We will also knockdown ATP release pathways and will overexpress nucleotide metabolizing activities to investigate the effect of ATP / Ado removal on mucin hydration activities in Th1- and CS-exposed HBE cells. Next (Aim 2), we will superimpose CB models on ATP release-deficient mice (Panx1 and Vnut double knockout mice) and CD39 (ATPase)-overexpressing mice to assess the effect of altered nucleotide homeostasis on mucus clearance/plugging in vivo. Lastly, (Aim 3) we will utilize samples and phenotype data from control subjects and CB/COPD patients (~1800 subjects) to: (i) investigate differences in nucleotide / nucleoside concentrations and nucleotidase activities in induced sputum between groups / subgroups and correlations to clinical measures of disease; and (ii) investigate potential mechanisms perturbing nucleotide homeostasis regulation utilizing gene expression in release and metabolic pathways known to modify nucleotide levels in ASL. Since the mechanisms leading to the production of hyperconcentrated mucus in CB/COPD are incompletely understood, completion of these studies will advance our understanding of the molecular and physiological processes by which ASL nucleotides regulate mucus hydration and protects against CB pathogenesis. This knowledge is essential for identifying novel disease biomarkers and therapeutic targets for mucus obstructive lung diseases.